PROJECT SUMMARY/ABSTRACT Mammalian tissues exhibit remarkable regenerative activity following injury, and defects in this process are associated with aging and degenerative diseases. Skeletal muscle regeneration requires a sustained proliferative response from muscle stem cells called satellite cells (SCs), yet it is unknown how this is achieved. Cellular proliferation is controlled by extracellular growth factors, or mitogens. The process of cellular proliferation, referred to as the cell cycle, involves cells exiting a non-proliferative G0 state and initiating the cell cycle in G1. At some point in G1, cells effectively escape the requirement for mitogens and commit to mitogen- independent completion of the cell cycle, but subsequent rounds of proliferation each require mitogen signaling during or just before G1. Insights into cell-cycle commitment mechanisms are primarily derived from in vitro studies of fibroblasts and immortalized epithelial cells, but it is unclear how relevant such systems are to the process of SC activation, where SCs exhibit a prolonged first G0/G1 phase, followed by multiple rounds of proliferation. Our preliminary work indicates that a mechanism of bistability, where a process remains indefinitely engaged despite the removal of mitogens, underlies cell-cycle commitment in the G0/G1 phase of SCs. The central hypothesis of this proposal is that a bistable G0/G1 mechanism underlies multiple rounds of mitogen-independent SC proliferation and effective muscle regeneration. The proposed work addresses the following specific aims: 1. Identify the mechanism of SC cell-cycle commitment through prolonged and multiple G0/G1 phases. 2. Establish mitogen signaling dynamics during muscle regeneration. 3. Determine the role of SC G0/G1 cell-cycle commitment during muscle regeneration. The mechanism of G0/G1 cell-cycle commitment will be identified using FACS-based SC isolation and ex vivo assays, including time-lapse imaging, immunofluorescence, and automated cell tracking and image analysis. In vivo mitogen signaling dynamics will be investigated using a muscle injury model and harvesting muscles for biochemical and transcriptomic analysis of growth factor availability and SC mitogen signaling. The functional role of SC G0/G1 cell-cycle commitment mechanisms in muscle regeneration will be tested using SC-specific conditional knockout mice and rescue by ectopic introduction of mitogens. Stanford is a leader in stem cell and regenerative sciences and will provide me with the technologies and collaborative network to develop into an independent research scientist. This research will uncover the fundamental relationship between mitogens and stem cell proliferation during muscle regeneration and may lead to therapeutic strategies for treating regenerative defects in aging and degenerative diseases.